Method for monitoring the effectiveness of teracycline in the treatment of asthma

ABSTRACT

The present invention is directed to a method of lowering excess IgE levels in a mammal suffering from a disease where IgE is pathogenic which method comprises administering to said mammal an IgE lowering effective amount of a tetracycline such as minocycline or doxycline. It is also directed to a method of monitoring the effectiveness of a drug in lowering the concentration of excess IgE in plasma in a mammal suffering from the disease in which IgE is pathogenic which method comprises making a first determination of the concentration of IgE in the plasma of said mammal at an initial time; administering to said mammal the drug; making a second determination of the concentration of IgE in the plasma of said mammal after the initial time and after administration of the drug and comparing the values obtained from the first and second determinations, such that if the value of the second determination is higher than or about the same as the value of the first determination and above a threshold level, then the dosage amount of the drug administered to the mammal is increased; otherwise the dosage amount is maintained.

BACKGROUND OF THE INVENTION

This invention relates to the discovery that minocycline and doxycline,and other tetracyclines are effective in lowering IgE levels in mammals,especially humans, suffering from a disease where IgE is pathogenic,such as allergies, asthma, especially human allergic response, anddiseases associated with an inflammatory response.

Diseases involving inflammation are characterized by the influx ofcertain cell types and mediators, the presence of which can lead totissue damage and sometimes death. Diseases involving inflammation areparticularly harmful when they afflict the respiratory system, resultingin obstructed breathing, hypoxemia, hypercapnia and lung tissue damage.Obstructive diseases of the airways are characterized by air flowlimitation (i.e., airflow obstruction or narrowing) due to constrictionof airway smooth muscle, edema and hypersecretion of mucous leading toincreased work in breathing, dyspnea, hypoxemia and hypercapnia.

A variety of inflamatory agents can provide air flow limitation, such asfor example, allergens. In particular, allergens and other agents inallergic or sensitized animals (i.e., antigens and haptens) cause therelease of inflammatory mediators that recruit cells involved ininflammation. Such cells include lymphocytes, eosinophils, mast cells,basophils, neutrophils, macrophages, monocytes, fibroblasts andplatelets. A variety of studies have linked the degree, severity andtiming of the inflammatory process with the degree of airwayhyperresponsiveness. Thus, a common consequence of inflammation isairflow limitation and/or airway hyperresponsiveness. Asthma is asignificant disease of the lung which is typically characterized byperiodic air flow limitation and/or hyperresponsiveness to variousstimuli which results in excessive airways narrowing. Othercharacteristics can include inflammation of airways and eosinophila.More particularly, allergic asthma is often characterized byeosinophilic airway inflammation arid airway responsiveness.

An estimated 16 million persons in the U.S. have asthma, which is about10% of the population. The numbers have increased about 25% in the last20 years. The estimated cost of treating asthma in the U.S. exceeds $6billion. About 25% of patients with asthma who seek emergency carerequire hospitalization. The largest single direct medical expenditurefor asthma has been in patient hospital services (emergency care), at acost of greater than $1.6 billion. The cost for prescription medicationsis at least $1.1 billion.

According to the National Ambulatory Medical Care Survey, asthmaaccounts for 1% of all ambulatory care visits and the disease continuesto be a significant cause of missed school days in children. Despiteimproved understanding of the disease process and better drugs, asthmamorbidity and mortality continues to rise in this country and worldwide.Thus, asthma constitutes a significant public health problem.

The pathophysiologic processes that attend the onset of an asthmaticepisode can be broken down into essentially two phases, both marked bybronchioconstriction, that causes wheezing, chest tightness, anddyspnea. The first, early phase asthmatic response is triggered byallergens and irritants. Allergens cross-link immunoglobulin (IgE)molecules bound to receptors on mast cells, causing them to release anumber of pre-formed inflammatory mediators, including histamine.Additional triggers include the osmotic changes in airway tissuesfollowing exercise and/or the inhalation of cold, dry air. The second,late phase response that follows is characterized by infiltration ofactivated eosinophilis and other inflammatory cells into airway tissues,epithelial desquamonon and by the presence of highly viscous mucuswithin the airway. The damage caused by this inflammatory responseleaves the airways “primed” or sensitized, such that smaller triggersare required to elicit subsequent asthma symptoms.

For instance, human allergic asthma, a disease characterized by airwayhyperresponsiveness and bronchial inflammation, is mediated by a varietyof activated leukocytes, including eosinophils, mast cells, CD4+Tlymphocytes, and CD 19+β cells.

Current treatments, which improve airway hyperresponsivenss, includevarious anti-inflammatory agents, which reduce mucosal inflammation andasthma pathogenesis; however their efficacies vary markedly.

Short acting β₂-adrenegric agonists, terbutaline and albuterol, long themainstay of asthma treatment, act primarily during the early phase asbronchodilators. The newer long acting β₂ agonists do not possesssignificant anti-inflammatory activity; they have no effect on bronchialhyperreactivity.

Numerous other drugs target specific aspects of the early or lateasthmatic responses. For example, antihistamines, like loratadine,inhibit early histamine-mediated inflammatory responses. Otherantihistamines, such as azelastine and ketotifen, have bothanti-inflammatory and weak bronchodilatory effects, but they currentlydo not have any established efficacy in asthma treatment.

Phosphodiesterase inhibitors, like theophylline/xanthines, may attenuatelate inflammatory responses, but there is no evidence that the compoundsdecrease bronchial hyperreactivity. Anticholinergics, like ipratopiumbromide, which are used in cases of acute asthma to inhibit severebronchoconstruction, have no effect on early or late phase inflammation,no effect on bronchial hyperreactivity and therefore essentially have norole in chronic therapy.

The corticosteroid drugs, like budesonide, are among the most potentanti-inflammatory agents. Inflammatory mediators or release inhibitors,like cromolyn and nedocromil, act by stabilizing mast cells andinhibiting the late phase inflammatory response to allergen. Thus,cromolyn and nedocromil, as well as the corticosteroids, all reducebronchial hyperactivity by minimizing the sensitivity effect ofinflammatory damage to the airways. These anti-inflammatory agents,however, do not produce bronchodilation.

Thus, while numerous drugs are currently available for the treatment ofasthma, these compounds are primarily palliative and/or have significantside effects. Moreover, allergen immunotherapy for asthma is limited inefficacy because patients frequently have sensitivities to multipleallergens and can experience immediate hypersensitivity reactions, withresultant decreased adherence to immunotherapy protocols.

Unfortunately, none of the aforementioned drugs target the underlyingcause of asthma. Consequently, new therapeutic approaches which targetthe underlying cause rather than the cascade of symptoms would be highlydesirable. The present inventors have searched for the underlying causeof asthmas, especially human allergic asthma. In asthma, CD4+ T cellssecrete IL-4, a (Th-)2 type cytokine, which is required for IgEproduction and which is implicated in airway hyperresponsiveness, aswell another cyctokines which increase IgE production. Although acorrelation between IgE and airway hyperresponsiveness has been allegedin allergic asthma, a cause and effect relationship has not as yet beenestablished. The present inventors have found that causal relationshipand have found that the tetracyclines, such as minocycline anddoxycylcine, and the like, suppress the excess concentration of IgE inthe blood plasma of patients suffering from human allergic asthma

An aspect of the present invention is directed to the use of minocyclineand doxycycline for treatment of asthma.

Both minocycline and doxycycline are known compounds. For example, ithas been reported that allergic steroid dependent asthmatic patientstreated with an oral administration of minocycline improved theirsymptoms (A.M. and P.M.), and decreased oral corticosteroid requirementsSee Joks et al., J. Allergy Clin. Immunol. 1998, 101:562. Additionalstudies of O'Dell,. et al. in Arthritis Rheum: 1997, 40: 842-848 andArthritis Rheum:, 1999, 42:1691-1695 have shown that treatment of mildand moderate rheumatoid arthritis (RA) patients with minocycline had noside effects, and appears to be an effective therapy for early RA.Moreover, Yu, et al. in Arthritis Rheum:, 1992, 35: 1150-1155 reportedthat treatment of dogs with minocycline or doxycycline reduced theseverity of osteoarthritis (OA), while studies by Thong, et al. in ClinExp Immunol., 1979, 35:443-446, have shown that doxycycline andtetracycline inhibit the ability of mice to mount delayed-typehypersensitivity responses. Further, studies in vitro have demonstratedthat treatment of human whole blood cultures with minocycline ortetracycline at physiological doses inhibits mitotic responses tophytohemagglutinin (See Ingham, et al., Antimicrob Chemother, 1991, 27:607-617) and inhibits inducible nitric oxide synthase (iNOS) expressionby murine macrophages See Amin, et al., PNAS, 1996, 93: 14014-14019.

The present inventors have found from their investigation a causalrelationship between excessive IgE levels and the airwayhyperresponsiveness found in asthma The present inventors-have foundthat drugs which lower excessive circulatory IgE levels in plasma areeffective in treating human allergic asthma. In particular the presentinventors have found that tetracyclines, such as minocycline anddoxycycline suppress excess IgE levels in patients suffering fromasthma, allergies, inflammatory conditions, or other diseases where IgEis pathogenic.

SUMMARY OF THE INVENTION

Thus, the present invention is directed to a method of lowering excessIgE concentration in the plasma of a mammal suffering from a diseasewhere IgE is pathogenic which method comprises administering to a mammalsuffering therefrom an IgE lowering effective amount of a tetracycline.In another embodiment, the present invention is directed to a method oflowering IgE concentration in the plasma of a mammal suffering from adisease in which IgE is pathogenic which comprises administering to saidmammal suffering therefrom an IgE lowering effective amount ofminocycline or doxycycline or a combination thereof Diseases in whichthe IgE is pathogenic include allergies and asthma, including humanallergic asthma and inflammatory conditions. The present inventors havealso found that patients, e.g., mammals, such as humans with higherconcentrations of IgE in their plasma are more prone to be sufferingform diseases where IgE is pathogenic, e.g., allergies and asthma,including human allergic asthma than those having a lower concentrationthereof. Thus, by monitoring the IgE concentration in the plasma, theeffectiveness of the drug administered to patients who suffer fromdiseases in which IgE is pathogenic, e.g., allergies, asthma, and thelike, which lower IgE concentration can be determined. Thus, anotheraspect of the present invention is directed to a method of monitoringthe effectiveness of a drug in lowering the concentration of IgE in theplasma in a mammal suffering from a disease in which IgE is pathogenicwhich method comprises making a first determination of the concentrationof IgE in the plasma at an initial time in said mammal; administering aneffective amount of drug which lowers IgE concentration in the plasma,e.g., a tetracycline, such as minocycline or doxycycline, or the like orcombination thereof to said mammal; at a second time subsequent to theinitial time, and after the administration of said drug, e.g., atetracycline, making a determination of the concentration of IgE in theplasma; comparing the values obtained from the first and seconddeterminations, such that if the value of the second determination ofthe free IgE level in the plasma is higher than or about the same as thefirst determination thereof and above a threshold level, then the dosageamount of the drug, e.g., the tetracycline, administered to the mammalis increased; otherwise the dosage regimen is maintained.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the IgE production in vitro by peripheral bloodmononuclear cells (PBMC) from asthmatic and non asthmatic subjects aftertetracycline treatment.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “patient” or “subject” refers to a warm bloodedanimal and preferably mammals, such as for example, cats, dogs, horse,cows, pigs, mice, rats and primates including humans. The preferredpatient is humans.

The term “drug” herein is used to connote a compound which is used tolower the IgE concentration in the plasma of a mammal, e.g., human. Thepreferred drugs are the tetracyclines.

The term “a tetracycline” or “tetracyclines” refers to thebroad-spectrum antibiotics which are members of the tetracycline family.Examples include tetracycline, rolitetracycline, oxytetracycline,chlortetracycline, demeclocycline, meclocycline, methacycline,doxycycline and minocycline, and the like. The preferred tetracyclinesare doxycycline and minocycline.

This terminology is to be distinguished from the antibiotic“tetracycline”. It is to be understood that when the term “tetracycline”is used herein by itself, that is, without an article (a, an, the) or ifused in the singular, it refers to the antibiotic tetracycline.

An embodiment of the present invention is directed to the use of IgElowering effective amounts of a tetracycline for the suppression ofelevated concentrations of IgE in the blood plasma of a patientsuffering from a disease in which IgE is pathogenic. In anotherembodiment, the present invention is directed to the use of IgE loweringeffective amounts of minocycline for the suppression of elevatedconcentrations of IgE in the blood plasma of the patient, e.g., a humansuffering from a disease in which IgE is pathogenic, especially humanallergic asthma. Another embodiment of the present invention is directedto the use of IgE lowering effective amounts of doxycycline for thesuppression of elevated concentrations of IgE in the blood plasma of thepatient, e.g., humans, suffering from a disease where IgE is pathogenicespecially human allergic asthma In a further embodiment, the presentinvention is directed to a use of a combination of both minocycline anddoxycycline in IgE lowering effective amounts for the suppression ofelevated concentrations of IgE in the blood plasma of a patients, e.g.,human suffering from disease where IgE is pathogenic, especially humanallergic asthma. Accordingly, minocycline or doxycycline or both incombination in amounts effective to lower the concentration of excessIgE concentrations in the plasma are useful for treating human allergicasthma. In addition, the tetracyclines, e.g. minocycline and doxycyclineare anti-inflammatory agents and can be used to treat inflammatoryconditions when administered to patients in IgE lowering effectiveamounts, as defined herein.

The tetracyclines are administered therefore in therapeuticallyeffective amounts.

The physician will determine the dosage of the tetracyclines which willbe most suitable and it will vary with the form of administration andthe particular compound chosen, and furthermore, it will vary dependingupon various factors, including but not limited to the patient undertreatment, the age of the patient, the severity of the condition beingtreated and the like. He will generally wish to initiate treatment withsmall dosages substantially less than the optimum dose of the compoundand increase the dosage by small increments until the optimum effectunder the circumstances is reached. The tetracyclines, when givenorally, are administered in dosages ranging from about 1 to about 400mg/day and more preferably from about 1 to about 300 mg/day. When givenparenterally, the tetracyclines are administered preferably in dosagesof, for example, about 1.5 to about 400 mg/day, and more preferably fromabout 1 to about 300 mg/day also depending upon the host and theseverity of the condition being treated and the compound utilized.

This dosage regimen may be adjusted by the physician to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The tetracyclines may be administered in a convenient manner, such as byoral, intravenous (where water soluble), intramuscular or subcutaneousroutes.

The tetracyclines may be orally administered, for example, with an inertdiluent or with an assimilable edible carrier, or it may be enclosed inhard or soft shell gelatin capsules, or it may be compressed intotablets, or it may be incorporated directly into the food of the diet.For oral therapeutic administration, the tetracyclines may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least1% of the appropriate tetracyclines. The percentage of the compositionsand preparations may, of course, be varied and may conveniently bebetween about 5 to about 80% of the weight of the unit. The amount ofthe tetracyclines used in such therapeutic compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention contain between about 25 mg and about1000 mg of a tetracycline including those containing about 25 mg, about50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, and about300 mg.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier.

Various other materials may be present as coatings or otherwise modifythe physical form of the dosage unit. For instance, tablets, pills, orcapsules may be coated with shellac, sugar or both. A syrup or elixirmay contain the tetracyclines, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any dosage unitform should be pharmaceutically pure and substantially non-toxic in theamounts employed. In addition, the tetracyclines may be incorporatedinto sustained-release preparations and formulations. For example,sustained release dosage forms are contemplated wherein thetetracyclines are bound to an ion exchange resin which, optionally, canbe coated with a diffusion barrier coating to modify the releaseproperties of the resin or wherein the tetracyclines are associated witha sustained release polymer known in the art, such ashydroxypropylmethylcellulose and the like.

The tetracyclines may also be administered parenterally orintraperitoneally. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating thetetracyclines in the required amount in the appropriate solvent with anyof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized tetracyclines into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders, the above solutions are vacuum dried or freeze-dried, asnecessary.

The tetracyclines can also be formulated and administered to the patientin solid or liquid particulate form by direct administration, e.g.,inhalation, into the respiratory system.

Solid or liquid particulate forms of the tetracyclines prepared forpracticing the present invention include particles of respirable size:that is, particles of a size sufficiently small to pass through themouth and larynx upon inhalation and into the bronchi and alveoli of thelungs. In general, particles ranging from about 1 to 10 microns in sizeare within the respirable range. The pharmaceutical compositionscontaining the tetracyclines are preferably administered by directinhalation into the respiratory system for delivery as a mist or otheraerosol or dry powder. Particles of non-respirable size which areincluded in the aerosol tend to be deposited in the throat andswallowed; thus the quantity of non-respirable particles in the aerosolis preferably minimized.

In the manufacture of the preferred local formulation, in accordancewith the description herein, the tetracylines are typically admixedwith, inter alia an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation.

Aerosols of liquid particles comprising the tetracyclines may beproduced by any suitable means, such as inhalatory delivery systems. Oneis a traditional nebulizer which works in a mechanism similar to thefamiliar perfume atomizer. The airborne particles are generated by a jetof air from either a compressor or compressed gas cylinder-passingthrough the device (pressure driven aerosol nebulizer). In addition,newer forms utilize an ultrasonic nebulizer by vibrating the liquid at aspeed of up to about 1 MHz. See, e.g., U.S. Pat. No. 4,501,729, thecontents of which are incorporated by reference. Nebulizers arecommercially available devices which transform solutions or suspensionsof the tetracyclines into a pharmaceutical aerosol mist either by meansof acceleration of compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers consist of the tetracyclines in aliquid carrier. The carrier is typically water (and most preferablysterile, pyrogen-free water) or a dilute aqueous alcoholic solution,preferably made isotonic but may be hypertonic with body fluids by theaddition of, for example, sodium chloride. Optional additives includepreservatives if the formulation is not made sterile, for example,methyl hydroxybenzoate, as well as antioxidants, flavoring agents,volatile oils, buffering agents and surfactants, which are normally usedin the preparation of pharmaceutical compositions.

Aerosols of solid particles comprising the tetracyclines may likewise beproduced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing a predetermined metered dose ofa medicament at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which may be delivered by means of aninsufflator or taken into the nasal cavity in the manner of a snuff. Inthe insufflator, the powder (e.g., a metered dose thereof effective tocarry out the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of thetetracycline or of a powder blend comprising the tetracycline, asuitable powder diluent, such as lactose, and an optional surfactant. Asecond type of illustrative aerosol generator comprises a metered doseinhaler.

Metered dose inhalers are pressurized aerosol dispensers, typicallycontaining a suspension or solution formulation of the tetracycline in aliquefied propellant. During use, these devices discharge theformulation through a valve, adapted to deliver a metered volume, fromabout 10 to about 22 microliters to produce a fine particle spraycontaining tetracycline.

Any propellant may be used in carrying out the present invention,including both chlorofluorocarbon-containing propellants andnon-chlorofluorocarbon-containing propellants. Fluorocarbon aerosolpropellants that may be employed in carrying out the present inventionincluding fluorocarbon propellants in which all hydrogens are replacedwith fluorine, chlorofluorocarbon propellants in which all hydrogens arereplaced with chlorine and at least one fluorine, hydrogen-containingfluorocarbon propellants, and hydrogen-containing chlorofluorocarbonpropellants. Examples of such propellants include, but are not limitedto: CF₃CHFCF₂, CF₃CH₂CF₂H, CF₃CHFCF₃, CF₃CH₂CF₃, CF₃CHCl—CF₂Cl,CF₃CHCl—CF₃, CF₃CHCl—CH₂Cl, CF₃CHF—CF₂Cl, and the like. A stabilizersuch as a fluoropolymer may optionally be included in formulations offluorocarbon propellants, such as described in U.S. Pat. No. 5,376,359to Johnson. The aerosol formulation may additionally contain one or moreco-solvents, for example, ethanol, surfactants, such as oleic acid orsorbitan trioleate, antioxidants and suitable flavoring agents.

Compositions containing respirable dry particles of micronizedtetracyclines may be prepared by grinding the dry active compound, withe.g., a mortar and pestle or other appropriate grinding device, and thenpassing the micronized composition through a 400 mesh screen to break upor separate out large agglomerates.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute. Aerosols containing greater amounts of thetetracycline may be administered more rapidly.

Typically, each aerosol may be delivered to the patient for a periodfrom about 30 seconds to about 20 minutes, with a delivery period ofabout 1 to 5 minutes being preferred.

The particulate composition comprising the tetracyclines may optionallycontain a carrier which serves to facilitate the formation of anaerosol. A suitable carrier is lactose, which may be blended with thetetracycline in any suitable ratio.

The tetracyclines are administered by this mode of administration intherapeutically effective amounts, as defined herein which amounts aredetermined by the physician.

The tetracyclines can also be applied in therapeutic effective amountsthrough a transdermal patch using techniques known to one of ordinaryskill in the art.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents for pharmaceuticalactive substances well known in the art. Except insofar as anyconventional media or agent is incompatible with the tetracyclines,their use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The tetracycline is compounded for convenient and effectiveadministration in effective amounts with a suitable pharmaceuticallyacceptable carrier in dosage unit form as hereinbefore described. A unitdosage, for example, contains the principal active cormpound in amountsranging from about 50 mg to about 1000 mg. If placed in solution, theconcentration of the tetracycline preferably ranges from about 10 mg/mlto about 250 mg/ml. The preferred mode of administration is oral. Thepresent inventors, have noted that in normal humans, not suffering fromasthma or allergies or diseases wherein IgE is pathogenic, theadministration of these tetracyclines does not increase theconcentration of IgE in the plasma

The tetracyclines are useful in treating asthma The tetracyclines areanti-inflammatory agents and can be used to treat inflammatoryconditions.

The present inventors have found that the higher the concentration ofthe free IgE in the plasma in those patients suffering from a diseasewhere IgE is pathogenic, the greater is the risk of the disease statebecoming aggravated or worsening. By lowering the amount of the free IgEin the plasma of those patients, the physician will lower the risk ofthe disease state becoming worse or becoming exacerbated.

The effectiveness of the tetracyclines in lowering the concentration ofthe excess IgE in the plasma can be monitored. For example, the IgElevels in the plasma of the blood, e.g., in peripheral blood, such asperipheral blood mononuclear cells (PBMC), of a patient suffering from adisease wherein IgE is pathogenic, e.g., allergies or asthma especiallyallergic asthma, e.g., human allergic asthma, inflammatory conditionsand the like can be measured prior to treatment. After a period oftreatment, the IgE concentration in the plasma is measured again and thechange in the concentration of IgE, if any, is noted. If theconcentration of the IgE in the plasma is above a threshold leveldetermined by the physician and is not decreased, then the physicianshould change the treatment regimen and prescribe a greater dose oftetracyclines for the treatment of the disease. If, on the other hand,the concentration of the free IgE is significantly lowered or is lessthan the threshold level, then the disease is under control, the risk ofthe disease state worsening is minimized, and the treatment regimen ismaintained. In a preferred embodiment, the threshold level is less thanabout 100 IU/ml and more preferably less than about 75 IU/ml and mostpreferably less than about 50 IU/ml.

The present inventors have noted that when a patient is suffering fromthe disease in which IgE is pathogenic, e.g., allergy or asthma, andwhen the IgE concentration in the serum is greater than 100 IU/ml, thereis a substantial risk of the disease worsening. On the other hand, ifthe IgE concentration in the plasma serum of this patient is less thanabout 100 IU/ml and more preferably less than about 75 IU/ml aftertreatment, then there is a lower risk that the disease will worsen.

Thus, using this techniques, the IgE levels of the patient is monitoredperiodically, e.g., at least about every-three to six months todetermine the free IgE level in the plasma and the regimen of treatmentreviewed; as indicated hereinabove, if the free concentration of the IgEincreases or is above the threshold level, then the physician willincrease the dosage and/or change the treatment regimen.

In another embodiment, the present invention is directed to theprophylaxis of the disease wherein IgE is pathogenic from becoming moresevere, which method comprises administering to the patientprophylatically effective amount of the tetracyclines, e.g., minocyclineor doxycline or combination thereof. The effective amount in thisembodiment is determined by the physical usually, this amount the sameas the therapeutic effective amount discussed that describedhereinabove.

The tetracyclines can also be given to mammals not suffering fromasthma, or allergies or other diseases where IgE is pathogenic byadministering to the patient prophylactically effective amount of thetetracyclines, e.g., minocycline or doxycycline or combination thereof.The effective amounts can be determined by the physician. As indicatedhereinabove, the normal person has a free plasma concentration of IgE ofless than about 50 IU/ml. Thus, the administration of the aforementionedtetracyclines will prevent and/or retard the onset of allergy orallergic asthma and/or other disease where IgE is pathogenic. Inaddition, it will prevent or lower the risk of the persons having a freeIgE concentration in the plasma increasing to the level where the personis at a substantial risk of suffering from a disease where IgE ispathogenic. Preferably, the free IgE level in the plasma, after suchtreatment, will remain less than about 50 IU/ml.

In the present specification, unless indicated to the contrary, theplural will connote the singular and, vice versa.

The term “treating”, “treat” or “treatment”, as used herein refers tothe reduction and/or alleviation of at least one adverse effect orsymptom of a disease where IgE is pathogenic. It refers to themanagement and care of a mammalian subject, preferably humans, for thepurpose of combating the disease, conditions or disorders where IgE ispathogenic, and includes the administration of the tetracyclines todelay the onset of at least one symptom or complication associated withthe disease, alleviating the symptom or effect or complicationsassociated therewith or in the alternative eliminating the disease orcondition.

The term “prophylaxis” or “prevent” or synonym thereto refers to theprevention or a measurable reduction in the likelihood of a patientacquiring a disease where IgE is pathogenic, even if the mammal issuffering form another malady which debilitates it and makes it moresusceptible to such a disease. If a patient or mammal is suffering froma disease where IgE is pathogenic, the term also refers to the reductionin the likelihood of the disease becoming acerbated.

The term “therapeutically effective amount” is synomous with “IgElowering effective amounts” and refers to the amount effective oreliminating or alleviating or curing the symptoms associated with adisease or malady where IgE is pathogenic or alleviating or curing thedisease altogether.

The term “prophylatically effective amount” refers to the amounteffective in preventing or reducing the likelihood of a mammal, e.g.,patient from acquiring a disease in which IgE is pathogenic. It alsorefers to the amount effective in preventing a mammal afflicted with adisease or malady where IgE is pathogenic from worsening or becomingmore severe.

As indicated herein, these “amounts” can be determined by the physician;however, it is preferred that these amounts are the same.

The following examples further illustrate the invention.

EXAMPLES

Patient Specimens

Peripheral blood (40 ml total) was obtained from asthmatic patients(male and female patients, 26-54 yrs. old, mean age 40±14) (n=7) fromthe SUNY Downstate Asthma Center of Excellence, and non asthmaticcontrols (n=7). Asthmatic patients presented with clinically definedmild intermittent to severe persistent asthma, with elevated serum IgElevels (>100 IU/ml). Asthma severity was assessed in accordance withNational Institutes of Health (NIH) guidelines. Airflow limitation waspresent in all patients at the time of evaluation. Patients gaveinformed consent for the use of their blood samples for an experimentalstudy. Control, non asthmatic, subjects (male and female patients, agematched), were recruited from the hospital staff of SUNY DOWNSTATEMEDICAL CENTER who showed no evidence of asthma as identified by historyand normal total IgE levels (<50 IU//ml). The study was approved by theinstitutional review board of the SUNY Downstate Medical Center,Brooklyn, N.Y., and the procedures followed were in accordance withinstitutional guidelines involving human subjects.

Blood

For studies of serum immunoglobulins, blood was collected into red topMonoject tubes (Sherwood Medical, St. Louis, Mo.) and allowed to clotfor 30 min at room temperature. Tubes were rimmed and centrifuged at1000 rpm for 10 min. Sera were collected and stored at −20° C.

For studies of surface markers, blood was collected into ethylenediaminetetraacetic acid Monoject tubes (Sherwood Medical) and stored at roomtemperature for up to 2 hr when complete blood counts were performed(Sysmex, McGraw Park, Ill.); flow microfluorimetry studies (CoulterEpics XL/MCL) (Beckman Coulter, Miami, Fla.) were performed within 3 hr.

Determination of Cell Surface Markers

Antibodies

Mouse anti-human monoclonal antibodies (mAbs) directly conjugated tofluoresce in isothiocyanate (FITC) CD45, Simultest CD3/CD4, SimultestCD3/CD8, Simultest CD3/CD19, and appropriately matched isotype controlmAbs, were purchased from BD Biosciences (San Jose, Calif.), and usedaccording to manufacturer's recommendation.

Immunoflourescence Assay

Peripheral blood (100 μl) was incubated with conjugated antibodies for10 min at room temperature. Erythrocytes were lysed with whole bloodlysing solution (limunoprep) (Beckman Coulter). Flow cytometric analysiswas performed on a Coulter Epics XL/MCL Flow Cytometer using System IIsoftware (Coulter) and CytoComp (Coulter). Forward and side scatter wereused to identify the lymphocyte population and CD45 was used toestablish an optimal lymphocyte gate. A minimum of 15,000 events werecollected. The gain on the photomultiplier tube detecting fluorescenceintensity was adjusted so that 99% of cells with background fluorescencestaining were scored between 10⁰ and 10¹ on a 4-decade log scale.Specific fluorescence was reported as the percentage of cells withrelative fluorescence intensity scored above background. Total numbersof cells were calculated from the lymphocyte count. Data-are expressedas total lymphocytes/mm³.

Serum Immunoglobulins

Serum immunoglobulins (IgM, IgG, and IgA) were determined bynephelometry which was performed, according to manufacturer'srecommendation (Beckman Array System, Beckman Coulter, Inc.) at theClinical Immunochemistry Laboratory at SUNY Downstate Medical Center.The results are expressed in mg/dL. (reference range for healthy adultserum: IgM: 60-263 mg/dL; IgG: 694-161 3 mg/dL; IgA: 69-378 mg/dL) SerumIgE levels were detected by the UniCAP Total IgE Fluoroenzymeimmunoassay (Pharmacia & Upjohn Diagnostics, Kalamazoo, Mich.) which wasperformed according to manufacturer's recommendation. To evaluate thetest results, the response for the patient's sample was compareddirectly to the response of the IgE calibrators. Data are expressed asIU/ml (reference range for healthy adult serum: IgE: 20-100 IU/ml).

Cytokine Specific mRNA

RNA extraction and polymerase chain reaction (PCR). Total cellular RNA(2 μg/ml) was extracted from PBMC in accordance with the procedure ofChomczynski, et al. in Anal. Biochem. 1987, 162: 156-159, using TrizolReagent (GIBCO/BRL), according to manufacturer's recommendation. Pelletswere dissolved in TE buffer (10 mM Tris HCl (Sigma, St. Louis, Mo.), pH7.5, 1 mM EDTA, Sigma), and stored at −70° C. in a Bio-Freezer (FormaScientific, Marietta, Ohio). Expression of interieukin-2 (IL-2),interieukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10),interferon-gamma (IFN-gamma), interferon-alpha (IFN-alpha), and epsilonspecific mRNA was determined using the Advantage One-Step RT-PCR Kit(Clontech, Palo Alto, Calif.), according to manufacturer'srecommendation. PCR was conducted using primer pairs specific for IL-2,IL-4, IL-6, IL-10, IFN-gamma, IFN-alpha, and epsilon (Expected bandsizes: 305, 344, 628, 328, 427, 303, and 149 basepairs (bp),respectively). A beta-actin primer set was used as an internal positivecontrol. Negative controls, consisting of no mRNA, but addition ofprimers, were included in every experiment. The PCR amplicons wereseparated by electrophoresis in a 1.8% agarose (Seakem LE) gel (FMC,Rocldand, Me.), and visualized with ethidium bromide (Sigma).

Cell Cultures

PBMC were separated from blood on a Ficoll-Paque (Pharmacia, Piscataway,N.J.) gradient (density 1.077). The PBMC were carefully removed using atransfer pipette (VWR Scientific, San Francisco, Calif.). Cells werewashed twice in RPMI-1640 medium (GIBCO/BRL, Grand Island, N.Y.),resuspended in RPMI (3 ml), and counted on a hemocytometer (FisherScientific, Springfield, N.J.). Viability was >98%, as judged by trypanblue exclusion.

Studies of IgE responses induced in vitro were carried out according tothe method of Sampson and Buckley in J. Immunol 1981, 127: 829-834.Briefly, PBMC (1.5×10⁶ in 1 ml) were cultured +anti-CD40 mAb (1 ug/ml)(BD Pharmingen, San Diego, Calif.), ± recombinant human interleukin-4(rhIL-4) (200 U/ml) (Genzyme Corp., Boston, Mass.), in the presence orabsence of varying concentrations of either minocycline (LederleParenterals Inc., Carolina, Puerto Rico) or doxycycline (AmericanPharmaceutical Partner Inc., Schaumburg, Ill.), 0.1, 1.0, 10 ug/ml) for0, 3, and 10 days at 37° C. in complete medium in a humidified 4% CO₂atmosphere. Cell viability was >98%, as judged by trypan blue exclusion,on day 10.

Antibiotics were reconstituted in sterile dH₂O, according tomanufacturer's recommendations. Complete medium contained RPMI-1640(Fisher Scientific) and was supplemented withN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer (HEPES) (25mM) (Fisher Scientific), penicillin (100 U/ml) (GIBCO/BRL), streptomycin(100 ug/ml) (Eli Lilly and Co., Indianapolis, Ind.), L-glutamine (2 mM)(GIBCO/BRL), and 10% Fetal Calf Serum (FCS) (GIB CO/BRL). Theconcentration of rhIL-4 in culture was within 1 bog of physiologicrelevance (<100 U/ml), according to bioassay data provided by supplier.

For the in vitro quantitative determination of IgE content in cellculture supernatants, solid phase sandwich enzyme linked immunosorbentassays (ELISAs) were performed using IgE ELISA Test Kits (Abexon Trend,Ramsey, Minn.). All ELISAs were performed according to themanufacturer's recommended procedure. Specimens were analyzed induplicate and a standard curve was derived from known concentrations ofIgE. Plates were read using an automated microplate reader (ModelELX800) (Bio-Tek Instruments, Inc., Winooski, Vt.), with a 450-nmmeasurement filter. Optical densities were converted to IU/ml and/orng/ml. (1 IU=2.4 ng/IgE protein).

Statistical Analysis

Mixed model ANOVAs were used to compare distributions of lymphocytesubpopulations and serum immunogbobulin levels between asthmatic and nonasthmatic groups. The groups were compared on each variable. A P valueof <0.05 was considered statistically significant for all comparisons.Statistical analyses were performed using SPSS for Windows version 10.0software (Chicago, Ill.).

Results

1. Asthmatic Patients: Medical History and Clinical Features

All asthmatic patients (n=7) exhibited mild intermittent to severepersistent signs or symptoms (>2×/wk) of asthma. Treatment regimensincluded β-agonist inhalers, inhaled corticosteroids, and if necessary,oral steroids. Table I summarizes the medical history and clinicalfeatures of the asthmatic patient. TABLE I* ASTHMATIC PATIENTS: MEDICALHISTORY AND CLINICAL FEATURES Sex Asthma Signs and Positive Skin EczemaSerum IgE Patient (Age) Ethnicity Classification SymptomsPharmacotherapy Test History (IU/ml)** 1 F Hispanic Severe PersistentContinual Inhaled steroids^(a) (+) (−) 355 (46) 2 F African SeverePersistent Continual Prednisone (+) (−) 719 (35) American 10 mg poqd^(b) 3 M Caucasian Mild Intermittent >2×/wk Beta Agonist PRN^(c) (+)(+) 241 (34) 4 F African Mild Persistent >2×/wk Inhaled steroids (+) (−)209 (43) American 5 M Caucasian Mild Intermittent >2×/wk Beta AgonistPRN (+) (+) 346 (45) 6 F Hispanic Mild Persistent >2×/wk InhaledSteroids (+) (−) 192 (54) 7 F African Severe Persistent ContinualInhaled Steroids (+) (−) 510 (26) American*Diagnosis and management of asthma in accordance with NationalInstitutes of Health (NIH) guidelines, July 1997.**Serum IgE levels of asthmatics (367 ± 51 IU/ml) > non asthmatics (35 ±51 IU/ml) (P = 0.001); serum 1 gM, IgG and IgA levels in asthmatics (128± 19, 1352 ± 140, 309 ± 53 mg/dl, respectively) were similar to nonasthmatic controls (127 ± 19, 1223 ± 140, 191 ± 53 mg/dl, respectively).Data are expressed as mg/db or IU/ml + pooled standard error (SE).^(a)Fluticasone propionate 44 mcg inhalation aerosol: 2 puffs po tid:ter in die (three times daily).^(b)po: per os (by mouth); qd: quaque die (once daily);^(c)PRN: pro re nata (when necessary).

Clinical profiles of asthma in this study included: positive skin prickand intradermal test(s) (tree and grass pollens, ragweed pollen, dustmite, American cockroach, Alternaria, Cladosporium, or cat antigens),and/or eczema, and elevated serum IgE (>100 IU/ml) levels. Serum IgElevels were significantly increased in asthmatic (367 IU/ml+51,coefficient of variation [CV]=0.1), compared with control subjects (35IU/ml+51, CV=1.0; P=0.00 1). In contrast, serum 1 gM, IgG, and IgA(mg/dl) levels in asthmatic patients (128+19, 1352+140, 309+53 mg/dl,respectively) resembled those of controls (127+19, 1223+140, 191+53mg/dl, respectively). At the time of study, none of the patients weretreated with allergen immunotherapy.

2. Distributions of Blood Lymphocyte Subpopulations

Asthmatic and non asthmatic subjects (n=7 per group) had similar totalnumbers of blood CD3+CD4+ T cells (779/mm³+73, coefficient of variation(CV)=0.09 and 766+115, CV=0.15, respectively) and CD19+ B cells (239+35,CV=0.14 and 379+95/mm³, CV=0.25, respectively). The results aretabulated in Table II. TABLE II* DISTRIBUTIONS OF LYMPHOCYTESUBPOPULATIONS IN PERIPHERAL BLOOD OF ASTHMATIC AND NON ASTHMATIC HUMANSFluorescent cells Lymphocytes CD3+ CD4+ CD3+ CD8+ CD19+ Subject mm³ (%)mm³ (%) mm³ (%) CD4/CD8 Asthmatic 779 ± 73  45 ± 3 378 ± 66^(a) 21 ± 3239 ± 35 14 ± 2 2.06 Non 766 ± 115 36 ± 3 568 ± 53^(a) 28 ± 3 379 ± 9517 ± 3 1.35 Asthmatic*The distributions of lymphocyte subpopulations in peripheral blood ofasthmatic (n = 7) and non asthmatic (n = 7) humans were determined byflow microfluorimetry (Coulter Epics XL/MCL). Data are expressed as meantotal cells/mm³ or mean percentage (%) of positive cells ± standarderror (SE).^(a)Asthmatic < non asthmatic (P = 0.045).

The CD3+CD8+ T cell numbers were significantly decreased in the blood ofasthmatic patients (378+66/mm³; CV=0.17), compared with non asthmaticsubjects (568+53/mm³; CV=0.09; P=0.045). In contrast, the CD4/CD8 ratiowas not significantly different between the asthmatic and non asthmaticgroups (2.06 and 1.35, respectively).

3. Cytokine Expression by PBMC

On day 0, PBMC from asthmatic patients (17%) expressed IL-2 specificmRNA, compared with non asthmatic subjects (57%). In contrast, PBMC fromasthmatic patients (78%) expressed IL-10 specific mRNA, compared withnon asthmatic subjects (29%). Neither group expressed IFN-gamma mRNA.However, IL-4, IL-6, and IFN-alpha, and epsilon (data not shown)specific mRNA were similarly expressed by PBMC of both groups, as shownin Table III. TABLE III* SUMMARY OF CYTOKINE PRODUCTION BY PERIPHERALBLOOD MONONUCLEAR CELLS (PBMC) FROM ASTHMATIC AND NON ASTHMATIC HUMANSCytokines Th-1 Th-2 Subject IL-2 IFN-γ IL-4 IL-6 IL-10 IFN-α Asthmatic 1− − + − + + 2 − − + + + + 3 + − + + + + 4 − − − + − + 5 nt −  nt**nt + + 6 − − + + + + 7 − − + + − + Non Asthmatic 1 − − + − − + 2 − − + +− + 3 + − − − + + 4 + − + + + + 5 − + − − − + 6 + − − − − + 7 + − − + −+*Unfractionated PBMC from asthmatic and non asthmatic subjects wereevaluated for the presence (+) or absence (−) of Th1 type (IL-2, IFN-γ)cytokines, Th2 type (IL-4, IL-6, IL-10) cytokines, and IFN-α. Expressionof cytokine-specific mRNA production was determined by AdvantageOne-Step RT-PCR (Clontech), as described in methods.nt: not tested.**nt - not determined4. Effect of Minocycline or Doxycydline on IgE Responses Induced Invitro

When PBMC from either asthmatic or non asthmatic subjects were culturedwith anti-CD40 mAb and rhIL-4, virtually no IgE was detected in culturesupernatants on days 0 and 3 (<2.5 ng/ml). In contrast, when asthmaticPBMC (5 of 7 patients) were cultured with anti-CD40 mAb and rh-IL-4,high levels of IgE were detected in supernatants on day 10 (28 ng/ml±12)as shown in Table IV. TABLE IV* DOXYCYCLINE OR MINOCYCLINE INHIBITS IgEPRODUCTION BY STIMULATED PBMC IgE Additions (ng/ml) % InhibitionAnti-CD40 + rhIL-4  28 ± 12  0 Anti-CD40 + rhIL-4 + 0.1 ug doxycycline20 ± 9 29 Anti-CD40 + rhIL-4 + 1.0 ug doxycycline 14 ± 7 50 Anti-CD40 +rhIL-4 + 10.0 ug doxycycline  5 ± 2 83 Anti-CD40 + rhIL-4 + 0.1 ugminocycline 16 ± 5 43 Anti-CD40 + rhIL-4 + 1.0 ug minocycline 18 ± 7 36Anti-CD40 + rhIL-4 + 10.0 ug minocycline  5 ± 2 83*Supernatants were collected from 10 day cultured PBMC (1.5 × 10⁶/ml)stimulated with anti-CD40 mAb (1 ug/ml) plus rhIL-4 (200 U/ml) with orwithout varying concentrations (0.1, 1.0, 10 ug/ml, respectively) ofdoxycycline or minocycline. The supernatants were collected and IgElevels were measured by IgE ELISA Test Kit (Alexon Trend). Results arefrom 5 experiments, repeated 3×, using PBMC obtained from 5 asthmaticsubjects. Data are expressed# as ng/ml ± standard error (SE).% Inhibition was calculated as follows:% Inhibition = 1- (IgE production in each treatment group/IgE productionof PBMC stimulated with anti-CD4O mAb plus rhIL-4 without treatment) ×100 (%).

As shown by the data, IgE levels from 2 of 7 asthmatic patients, and allnon asthmatic patients did not increase (<2.5 ng/ml). No increases inIgE were detected when PBMC from either asthmatic or non asthmaticsubjects were cultured with either anti-CD40 mAb or rhTL-4 alone (<2.5ng/ml), or with either anti-CD40 mAb or rhIL-4 alone in the presence ofeither minocycline or doxycline (<2.5 ng/ml).

When either minocycline or doxycycline were included in cultures withasthmatic PBMC and anti-CD40 mnAb and rhIL-4, the high levels of IgEobtained on day 10 in the absence of these tetracycline were stronglysuppressed, in dose dependent fashion (to >80% with 10 ug/ml), as shownin Table IV. There was no change in IgE levels when either minocyclineor doxycycline was included in cultures of non asthmatic PBMC withanti-CD40 mnAb and rhIL-4 (<2.5 ng/ml), (See FIG. 1). More specifically,FIG. 1 illustrates the IgE production in vitro by PMC from asthma andnon asthmatic subjects after tetracycline treatment. Human PBMC(1.5×10⁶/1 ml) were obtained from asthmatic and non asthmatic subjects,and cultured ±anti-CD40 mAb (1 ug/ml)+rhIL-4 (200 U/ml), in the presenceor absence of minocycline (10 ug/ml) or doxycycline (10 ug/ml) for 10days. High levels of IgE obtained on day 10 in the absence oftetracyclines were strongly suppressed when tetracyclines were includedin cultures of PBMC from asthmatic patients. There was no change in IgElevels when tetracyclines were included in cultures of PBMC from nonasthmatic subjects. Results are from 12 experiments, repeated threetimes, using PBMC obtained from asthmatic subjects (n=5) and nonasthmatic subjects (n=7). Data are expressed as ng/ml ± standard error(SE).

As shown, minocycline and doxycycbine suppressed anti-CD40 mAb andrhIL-4 mediated induction of IgE responses by PBMC obtained fromasthmatic serum IgE+ atopic donors (asthmatic donors).

From the data, anti-CD40/rhIL-4 induced 5-10 fold higher IgE levels inculture supernatants of PBMC obtained from the asthmatic donors comparedwith PBMC of non asthmatic, non atopic serum IgE negative donors (nonasthmatic donors). This was true despite the fact that there weresimilar numbers of both CD19+ B cells and CD4+ T cells in cultures ofboth donor groups (see Table II) on day 0. However, PBMC of theasthmatic donors contained 20-30% fewer CD8+ T cells than those of thenon asthmatic donors, with a concomitant increase in other cells inPBMC, all of which have not as yet been identified. Without wishing tobe bound it is believed that they include gamma/delta T cells andnatural killer cells. It has been reported that gamma/delta T cellsdownregulate airway responsiveness to allergen challenge. Withoutwishing to be bound, it is believed that this is effected by controllingthe “repair” response of the airway epithelium to alpha/beta T cellmediated damage.

In asthma, both CD4 and CD8 T cell subsets are activated and theirnumbers are increased in the airways compared to non asthmatic subjects.It has been reported that CD8 T cells are critical in mediatingrespiratory syncitial virus (RSV)-induced development of lungeosinophilia and airway hyperresponsiveness following allergic airwaysensitization in mice. In animal models, it has been reported that CD8+T cells can attenuate allergic responses including IgE synthesis.Without wishing to be bound CD8+ T cells may provide suppressor functionby exerting their various cytokines or through regulation ofinflammatory mediators.

The results herein show that asthmatics may exhibit reduced CD8+ T cellsuppressor function, thus leading to the perpetuation of CD4+ Tcell-mediated inflammation and pro inflammatory (Th2) cytokines. It isbelieved, without wishing to be bound that the natural immune responseto inhaled protein antigens, especially in rats expressing the low IgEresponder phenotype, includes a MHC class I-restricted CD8+ T cellcomponent, which is associated with active suppression of IgE antibodyproduction.

The PBMC obtained from asthmatic donors also differed from non asthmaticdonors with respect to expression of constitutive IL-2 and-IL-10 mRNA,and in the levels of IgE they secreted in culture. IL-2 mRNA, which wasexpressed by PBMC of non asthmatic donors (57%) was expressed by fewerasthmatic patients (17%), whereas IL-10 specific mRNA was expressed inthe majority of asthmatic patients (78%), compared with non asthmaticdonors (29%), implicating the possibility of a reduced Th1-type responseand/or an increased Th2-type response in asthmatics. In general,constitutive cytokine specific mRNA expression for IL-4, IL-6,IFN-gamma, IFN-alpha, and epsilon by PBMC was similar for both groups(see Table III). Without wishing to be bound it is believed thatTh2-type cytokines play an important role in the immunological processesof allergic asthma. However, the differences in processing andexpression of these mRNA transcripts is unknown.

Without wishing to be bound, it is believed that the substantiallyhigher levels of IgE secreted by PBMC of the asthmatic group in responseto the nonspecific stimulus, anti-CD40/rhIL-4, show that differentregulatory cells such as CD4+ Th2 cells, and macrophages may be presentin the asthmatic compared with the non asthmatic PBMC. Although it iswell recognized that Th2-type cytokines (IL-4, IL-6, IL-10,respectively) favor IgE responses, and these asthmatic patients had highlevels of serum IgE at the time at which their PBMC were obtained (seeTable I), there were no detectable differences in mRNA expression of Th2vs. Th1-type cytokines by PBMC of the asthmatic vs. non asthmaticdonors, except for IL-2 and IL-10 mRNA expression. However, othercytokines may have been secreted in vitro to account for the dramaticincrease of IgE production by PBMC of the asthmatic group.

The significantly increased IgE production by asthmatics compared withnon asthmatic PBMC cannot, however, simply be attributed to increasednumbers of either CD19+ B cells, or CD4+ T cells, since similar numberswere present in PBMC of both groups. Without wishing to be bound, it isbelieved that IL-4 acts as an important inflammatory mediator in asthmaIL-4 is also required for induction of IgE responses in vitro.

It is also believed, without wishing to be bound, that another Th2-typecytokine, IL-13, similar to IL-4, can induce IgE synthesis by human Bcells. IL-13 induces IgE synthesis by PBMC as well as by purified Bcells obtained from PBMC in the presence of activated CD4+ T cellclones. In addition, IL-13 induces B-cell proliferation anddifferentiation into IgE-secreting cells in the presence of the ligandfor CD40, and is believed to contribute to the induction of IgEswitching and the maintenance of ongoing IgE synthesis in vivo.

As shown by the exemplification, minocycline and doxycycline suppressanti-CD40/rhIL-4 mediated induction of IgE responses by PBMC ofasthmatic donors, in dose dependent fashion, but had no effect on thelower IgE responses elicited by these agents from PBMC of the nonasthmatic donors (Figure I).

Thus, minocycline and doxycycline suppressed in vitro induction of IgEresponses. Other agents previously shown to suppress IgE responses bothin vivo and/or in vitro include: the neuropeptide, substance P, thebacterial cell wall products muramyldipeptide and murabutide, andcytokines such as IL-8, which inhibits IgE production, TGF-Beta, whichacts directly on B cells and targets epsilon gennline transcriptexpression, IFN-alpha, IFN-gamma, and IL-12, which also inhibit IgEsecretion.

Thus, in another embodiment of the present invention, these and/or otherIgE agents which lower IgE concentrations in the plasma can be used totreat patients suffering from diseases in which IgE is pathogenic andthese are administered in effective amounts as defined herein. Moreover,if these are administered to such patients, the effectiveness thereofcan be determined by monitoring the free IgE concentration in the plasmain the manner, as described hereinabove.

While the foregoing specification teaches the principle of the presentinvention, with examples provided for the purpose of illustration, theseteachings will make apparent to those skilled in the art otherembodiments and examples. These other embodiments and examples are alsowithin the scope of the present invention.

1. A method for reducing IgE concentrations in the blood of a patientsuffering from a disease comprising administering a therapeuticallyeffective amount of an antibiotic composition.
 2. The method of claim 1wherein said patient is suffering from allergic asthma.
 3. The method ofclaim 1 wherein said antibiotic is a tetracycline.
 4. The method ofclaim 3 wherein said antibiotic is selected from the group consisting oftetracycline, rolitetracycline, oxytetracycline, chlorotetracycline,democlocycline, meclocycline, methacycline, doxycycline and monocycline.5. The method of claim 3 wherein said antibiotic comprises tetracyclineor minocycline.
 6. The method of claim 4 wherein said antibioticcomprises a combination of tetracycline and minocycline.
 7. The methodof claim 4 wherein said antibiotic comprises doxycycline.
 8. The methodof claim 4 wherein said antibiotic comprises a combination ofdoxycycline and minocycline.
 9. The method of claim 1 wherein saidtherapeutically effective amount of antibiotic is administered orally.10. The method of claim 9 wherein said therapeutically effective amountof antibiotic is administered in an amount of about 1 to about 300mg/day.
 11. The method of claim 1 wherein said therapeutically effectiveamount of antibiotic is administered parenterally.
 12. The method ofclaim 11 wherein said therapeutically effective amount of antibiotic isadministered in an amount of about 1 to about 300 mg/day.
 13. The methodof claim 3 wherein said tetracycline is present in an amount betweenabout 25 mg and about 100 mg.
 14. The method of claim 1 wherein saidcomposition further comprises a pharmaceutically acceptable carrier. 15.A method of treating asthma in a patient comprising administering atherapeutically effective amount of an antibiotic composition.
 16. Themethod of claim 15 wherein said antibiotic is a tetracycline.
 17. Themethod of claim 16 wherein said antibiotic is selected from the groupconsisting of tetracycline, rolitetracycline, oxytetracycline,chlorotetracycline, democlocycline, meclocycline, methacycline,doxycycline and monocycline.
 18. The method of claim 16 wherein saidantibiotic comprises tetracycline or minocycline.
 19. The method ofclaim 17 wherein said antibiotic comprises a combination of tetracyclineand minocycline.
 20. The method of claim 17 wherein said antibioticcomprises a combination of doxycycline and minocycline.
 21. The methodof claim 15, wherein said therapeutically effective amount of antibioticis administered orally.
 22. The method of claim 21 wherein saidtherapeutically effective amount of antibiotic is administered in anamount of about 1 to about 300 mg/day.
 23. The method of claim 15wherein said therapeutically effective amount of antibiotic isadministered parenterally.
 24. The method of claim 23 wherein saidtherapeutically effective amount of antibiotic is administered in anamount of about 1 to about 300 mg/day.
 25. The method of claim 16wherein said tetracycline is present in an amount between about 25 mgand about 100 mg.
 26. The method of claim 15 wherein said compositionfurther comprises a pharmaceutically acceptable carrier.
 27. A method ofmonitoring the effectiveness of a drug in lowering the concentration ofIgE in the plasma of a mammal suffering from a disease comprising: a)making an initial determination of the concentration of IgE in theplasma at a first time in said mammal; b) administering an effectiveamount of a drug which lowers IgE concentration in the plasma; c) makinga determination of the concentration of IgE in the plasma at a timesubsequent to the initial determination; and d) comparing the valuesobtained from the first and second determination wherein if the value ofthe second determination of the free IgE level is higher than or aboutthe same as the first determination and above a threshold level, thenthe dosage amount of the drug is increased.
 28. The method of claim 27wherein said drug is a tetracycline.
 29. The method of claim 28 whereinsaid tetracycline is minocycline or doxycycline or a combinationthereof.